Energy transfer assay method and reagent

ABSTRACT

Disclosed is a non-fluorescent cyanine dye that may be used as an acceptor in fluorescence energy transfer assays involving the detection of binding and/or cleavage events in reactions involving biological molecules, and assay methods utilising such dyes. The non-fluorescent cyanine dye is a compound of formula (I), wherein the linker group Q contains at least one double bond and forms a conjugated system with the rings containing X and Y; groups R 3 , R 4 , R 5  and R 6  are attached to the rings containing X and Y, or optionally, are attached to atoms of the Z 1  and Z 2  ring structures; Z 1  and Z 2  each represent a bond or the atoms necessary to complete one or two fused aromatic rings each ring having five or six atoms, selected from carbon atoms and, optionally, no more than two oxygen, nitrogen and sulphur atoms; at least one of groups R 1 , R 2 , R 3 , R 4 , R 5 , R 6 and R 7  is a target bonding group; any remaining groups R 3 , R 4 , R 5 , R 6  and R 7  groups are independently selected from the group consisting of hydrogen, C 1 -C 4  alkyl, OR 9 , COOR 9 , nitro, amino, acylamino, quaternary ammonium, phosphate sulphonate and sulphate, where R 9  is selected from H and C 1 -C 4  alkyl; any remaining R 1  and R 2  are selected from C 1 -C 10  alkyl which may be unsubstituted or substituted with phenyl, the phenyl being optionally substituted by up to two substituents selected from carboxyl, sulphonate and nitro groups; characterised in that at least one of the groups R 1 , R 2 , R 3 , R 4 , R 5 , R 6  and R 7  comprises a substituent which reduces the fluorescence emission of said dye such that it is essentially non-fluorescent.

The present invention relates to the field of fluorescence resonanceenergy transfer. In particular, the invention relates to fluorogenicassays which include a novel class of non-fluorescent quenching dyes,and to novel quenching dye compounds thereof.

Fluorescence resonance energy transfer (FRET) occurs between theelectronic excited states of two fluorophores when they are insufficient proximity to each other, in which the excited-state energy ofthe donor fluorophore is transferred to the acceptor fluorophore. Theresult is a decrease in the lifetime and a quenching of fluorescence ofthe donor species and a concomitant increase in the fluorescenceintensity of the acceptor species. In one application of this principle,a fluorescent moiety is caused to be in close proximity to a quenchermolecule. In this configuration, the energy from the excited donorfluorophore is transferred to the quencher and dissipated as heat ratherthan fluorescence energy.

The use of fluorescence resonance energy transfer (FRET) labels inbiological systems is well known. The principle has been used in thedetection of binding events or cleavage reactions in assays which employfluorescence resonance energy transfer. In the case of peptide cleavagereactions, a fluorescent donor molecule and fluorescent acceptormolecule are attached to a peptide substrate on either side of thepeptide bond to be cleaved and at such a distance that non-radiativeenergy transfer between the donor and the acceptor species takes place.For example, EPA 428000 discloses a novel fluorogenic peptide substrateinvolving a fluorescent donor molecule and a quenching acceptor moleculeattached thereto. The labelled substrate can be used in the detectionand assay of a viral protease enzyme, whereby, if there is enzymepresent in a test sample, the substrate is cleaved and the iikonor andacceptor species are thereby separated. The resultant fluorescentemission of the donor species can be measured. Suitable fluorescentdonors include fluorescein derivatives, coumarins and5-((2-aminoethyl)amino)-naphthalene-1-sulphonic acid (EDANS). Suitablequenching acceptors include 2,4-dinitrophenyl (DNP) and4-(4-dimethylaminophenyl)azobenzoic acid (DABCYL).

Fluorescence energy transfer has also been used in the study of nucleicacid hybridisation. For example, Tyagi and Kramer (Nature Biotechnology,14, 303-8, (1996)) disclose homogeneous hybridisation assays whichutilise fluorescent labelled probes. The hair-pin probes comprise asingle-stranded nucleic acid sequence that is complementary to thetarget nucleic acid, together with a stem sequence formed from twocomplementary arms which flank the probe sequence. A fluorophore (EDANS)is attached to one arm and the non-fluorescent quencher moiety (DABCYL)is attached to the complementary arm. In the absence of target, the stemkeeps the fluorescent and quenching groups in close proximity causingthe fluorescence of the fluorophore to be quenched. When the probe isallowed to bind to a nucleic acid target, it undergoes a conformationalchange, forming a more stable hybrid with the target and forcing the armsequences (and the fluorophore and quencher) to move apart. Thefluorophore will then emit fluorescence when excited by light of asuitable wavelength.

The success of the fluorescence resonance energy transfer approach isdependent upon the choice of the appropriate donor/acceptor pair. Ifenergy transfer between the donor and acceptor can be optimised,residual fluorescence is minimised when the donor/quencher pair are inclose proximity and a large change in signal can be obtained when theyare separated. There is an increasing trend towards assayminiaturisation and in high throughput screening assays and, as aresult, it is beneficial to use fluorophores with high extinctioncoefficients in order to achieve the sensitivity levels required. Afurther problem associated with such assays is due to colour quenchingcaused by the presence in the assay medium of coloured samples whichtend to absorb strongly in the 350-450 nm region of the spectrum.

The present invention provides a non-fluorescent cyanine acceptor dyethat can be used as one component of a fluorescent donor/acceptor pairfor assays involving the detection of binding and/or cleavage events inreactions involving biological molecules. The fluorescent donor dyepossesses a high extinction coefficient, thereby enabling the detectionof low levels of the fluorophore. Moreover, the fluorescent dye pairhave excitation and emission wavelengths in a range which issubstantially free from auto-fluorescence associated with biologicalsamples and from quenching due to coloured samples. Additionally, thedyes are relatively pH insensitive and they possess a high degree ofspectral overlap, allowing efficient energy transfer.

Accordingly, the present invention relates to a compound of formula (1):

wherein the linker group Q contains at least one double bond and forms aconjugated system with the rings containing X and Y;

groups R³, R⁴, R⁵ and R⁶ are attached to the rings containing X and Y,or optionally, are attached to atoms of the Z¹ and Z² ring structures;

Z¹ and Z² each represent a bond or the atoms necessary to complete oneor two fused aromatic rings each ring having five or six atoms, selectedfrom carbon atoms and, optionally, no more than two oxygen, nitrogen andsulphur atoms;

X and Y are the same or different and are selected from bis-C₁-C₄ alkyl-and C₄-C₅ spiro alkyl-substituted carbon, oxygen, sulphur, selenium,—CH═CH— and N—W wherein N is nitrogen and W is selected from hydrogen, agroup —(CH₂)_(m)R⁸ where m is an integer from 1 to 26 and R⁸ is selectedfrom hydrogen, amino, aldehyde, acetal, ketal, halo, cyano, aryl,heteroaryl, hydroxyl, sulphonate, sulphate, carboxylate, substitutedamino, quaternary ammonium, nitro, primary amide, substituted amide.,and groups reactive with amino, hydroxyl, carbonyl, carboxyl,phosphoryl, and sulphydryl groups;

at least one of groups R¹, R², R³, R⁴, R⁵, R⁶ and R⁷ is a target bondinggroup;

any remaining groups R³, R⁴, R⁵, R⁶ and R⁷ groups are independentlyselected from the group consisting of hydrogen, C₁-C₄ alkyl, OR⁹, COOR⁹,nitro, amino, acylamino, quaternary ammonium, phosphate, suiphonate andsulphate, where R⁹ is selected from H and C₁-C₄ alkyl;

any remaining R¹ and R² are selected from C₁-C₁₀ alkyl which may beunsubstituted or substituted with phenyl the phenyl being optionallysubstituted by up to two substituents selected from carboxyl, sulphonateand nitro groups;

characterised in that at least one of the groups R¹, R², R³, R⁴, R⁶, R⁶and R⁷ comprises a substituent which reduces the fluorescence emissionof said dye such that it is essentially non-fluorescent;

provided that the linker group Q is not a squaraine ring system.

Preferably the linker group Q contains 1, 2 or 3 double bonds inconjugation with the rings containing X and Y.

Preferably Q is the group:

wherein the groups R¹⁰ are selected from hydrogen and C₁-C₄ alkyl whichmay be unsubstituted or substituted with phenyl, or two or more of R¹⁰together with the group:

form a hydrocarbon ring system substituted with R⁷ and which mayoptionally contain a heteroatom selected from —O—, —S—, or >NR⁷, whereinR⁷ is hereinbefore defined; and

n=1, 2 or 3.

Suitably, the non-fluorescent cyanine dye for use in the presentinvention is a compound having the formula (2):

wherein groups R³, R⁴, R⁵ and R⁶ are attached to the rings containing Xand Y or, optionally, are attached to atoms of the Z¹ and Z² ringstructures and n is an integer from 1-3;

Z¹ and Z² each represent a bond or the atoms necessary to complete oneor two fused aromatic rings each ring having five or six atoms, selectedfrom carbon atoms and, optionally, no more than two oxygen, nitrogen andsulphur atoms;

X and Y are the same or different and are selected from bis-C₁-C₄alkyl-and C₄-C₅ spiro alkyl-substituted carbon, oxygen, sulphur,selenium —CH=CH— and N—W wherein N is nitrogen and W is selected fromhydrogen, a group —(CH₂)mR⁸ where m is an integer from 1 to 26 and R⁸ isselected from hydrogen, amino, aldehyde, acetal, ketal, halo, cyano,aryl, heteroaryl, hydroxyl, sulphonate, sulphate, carboxylate,substituted amino, quaternary ammonium, nitro, primary amide,substituted amide, and groups reactive with amino, hydroxyl, carbonyl,carboxyl, phosphoryl, and sulphydryl groups;

at least one of groups R¹, R², R³, R⁴, R⁵, R⁶ and R⁷ is a target bondinggroup;

any remaining groups R³, R⁴, R⁵, R⁶ and R⁷ groups are independentlyselected from the group consisting of hydrogen, C₁-C₄ alkyl, OR⁹, COOR⁹,

nitro, amino, acylamino, quaternary ammonium, phosphate, sulphonate andsulphate, where R⁹ is selected from H and C₁-C₄ alkyl;

any remaining R¹ and R² are selected from C₁-C₁₀ alkyl which may beunsubstituted or substituted with phenyl the phenyl being optionallysubstituted by up to two substituents selected from carboxyl, sulphonateand nitro groups;

characterised in that at least one of the groups R¹, R², R³, R⁴, R⁵, R⁶and R⁷ comprises a substituent which reduces the fluorescence emissionof said dye such that it is essentially non-fluorescent.

Suitably at least one of the groups R¹, R², R³, R⁴, R⁵, R⁶ and R⁷of thedyes according to the present invention comprises a substituent whichreduces the fluorescence emission of the dye such that it is essentiallynon-fluorescent. Suitably, at least one of groups R³, R⁴, R⁵, R⁶ and R⁷of the non-fluorescent cyanine dyes of structures (1) and (2) is a nitrogroup which may be attached directly to the rings containing X and Y. Inthe alternative, a mono- or di-nitro-substituted benzyl group may beattached to the rings containing X and Y, which optionally may befurther substituted with one or more nitro groups attached directly tothe aromatic rings. Preferably, at least one of groups R¹, R², R³, R⁴,R⁵, R⁶ and R⁷ of the non-fluorescent cyanine dyes of structures (1) and(2) comprises at least one nitro group.

The target bonding group R¹, R², R³, R⁴, R⁵, R⁶ and R⁷ can be any groupsuitable for attaching the non-fluorescent cyanine dye to a targetmaterial, such as a carrier material or a biological compound and assuch will be well known to those skilled in the art. For example, thetarget bonding group may be a reactive group for reacting with afunctional group on the target material. Alternatively, the targetbonding group may be a functional group and the target may contain thereactive constituent.

Preferably, the target bonding group is of the structure—E—F where E isa spacer group and F is the reactive or functional group. A reactivegroup of the dye can react under suitable conditions with a functionalgroup of a target molecule; a functional group of the dye can reactunder suitable conditions with a reactive group of the target molecule,whereby the target molecule becomes labelled with the dye.

Preferably, the reactive group F is selected from carboxyl, succinimidylester, sulpho-succinimidyl ester, isothiocyanate, maleimide,haloacetamide, acid halide, hydrazide, vinylsulphone, dichlorotriazineand phosphoramidite. Preferably, the functional group F is selected fromhydroxy, amino, sulphydryl, imidazole, carbonyl including aldehyde andketone, phosphate and thiophosphate. By virtue of these reactive andfunctional groups the non-fluorescent cyanine dye may be reacted withand covalently bound to target materials.

Suitable spacer groups may contain 1-60 chain atoms selected from thegroup consisting of carbon, nitrogen, oxygen, sulphur and phosphorus.For example the spacer group may be:

—(CHR′)_(p)—

—{(CHR′)_(q)—O—(CHR′)_(r)—}_(s)—

—{(CHR′)_(q)—NR′—(CHR′)_(r)}_(s)—

—{(CHR′)_(q)—(CH═CH)—(CHR′)_(r)}_(s)—

—{(CHR′)_(q)—Ar—(CHR′)_(r)—}_(s)—

—{(CHR′)_(q)—CO—NR′—(CHR′)_(r)—}_(s)—

—{(CHR′)_(q)—CO—Ar—NR′—(CHR′)_(r)—}_(s)—

where R′ is hydrogen, or C₁-C₄ alkyl which may be optionally substitutedwith suiphonate, Ar is phenylene, optionally substituted withsulphonate, ρ is 1-20, preferably 1-10, q is 1-5, r is 0-5 and s is 1-5.

Specific examples of reactive groups R¹-R⁷ and the groups with whichR¹-R⁷ can react are provided in Table 1. In the alternative, the R¹-R⁷may be the functional groups of Table 1 which would react with thereactive groups of a target molecule.

TABLE 1 Possible Reactive Substituents and Functional Groups ReactiveTherewith Reactive Groups Functional Groups succinimidyl esters primaryamino, secondary amino anhydrides, acid halides primary amino, secondaryamino, hydroxyl isothiocyanate amino groups vinylsulphone amino groupsdichlorotriazines amino groups haloacetamides, maleimides thiols,imidazoles, hydroxyl, amines carboxyl amino, hydroxyl, thiolsphosphoramidites hydroxyl groups

Particularly suitable reactive groups R¹-R⁷ which are especially usefulfor labelling target components with available amino and hydroxylfunctional groups include:

where m is an integer from 1-10.

Aryl is an aromatic substituent containing one or two fused aromaticrings containing 6 to 10 carbon atoms, for example phenyl or naphthyl,the aryl being optionally and independently substituted by one or moresubstituents, for example halogen, straight or branched chain alkylgroups containing 1 to 10 carbon atoms, aralkyl and alkoxy for examplemethoxy, ethoxy, propoxy and n-butoxy.

Heteroaryl is a mono- or bicyclic 5 to 10 membered aromatic ring systemcontaining at least one and no more than 3 heteroatoms which may beselected from N, O, and S and is optionally and independentlysubstituted by one or more substituents, for example halogen, straightor branched chain alkyl groups containing 1 to 10 carbon atoms, aralkyland alkoxy for example methoxy, ethoxy, propoxy and n-butoxy.

Aralkyl is a C₁-C₆ alkyl group substituted by an aryf or heteroarylgroup.

Halogen and halo groups are selected from fluorine, chlorine, bromineand iodine.

The non-fluorescent cyanine dyes for use in the present invention mayalso include water solubilising constituents attached thereto forconferring a hydrophilic characteristic to the dye. They may be attacheddirectly to the aromatic ring system of the cyanine dye or they may beattached to the spacer group E. Suitable solubilising constituents maybe selected from the group consisting of sulphonate, sulphate,phosphonate, phosphate, quaternary ammonium and hydroxyl. Sulphonate orsulphonic acid groups attached directly to the aromatic ring of thenon-fluorescent quenching dye are to be particularly preferred. Watersolubility may be necessary when labelling proteins.

In a second aspect, the present invention relates to a biologicalmaterial labelled with a non-fluorescent cyanine dye.

In a further aspect, the invention relates to a biological materialwhich comprises two components one of which is labelled with afluorescent dye which may act as a donor of resonance energy and theother with a non-fluorescent cyanine dye which may act as an acceptor ofresonance energy transferred from the donor.

In a still further aspect, the invention relates to an assay methodwhich comprises:

i) separating two components which are in an energy transferrelationship, the first component being labelled with a fluorescentdonor dye and the second component being labelled with a non-fluorescentcyanine acceptor dye; and,

ii) detecting the presence of the first component by measuring emittedfluorescence.

In a still further aspect the invention relates to an assay method whichcomprises:

i) binding one component of a specific binding pair with a secondcomponent of said pair, said first component being labelled with afluorescent donor dye and said second component being labelled with anon-fluorescent cyanine acceptor dye to bring about an energy transferrelationship between said first and second components; and,

ii) detecting the binding of the first and second components bymeasuring emitted fluorescence.

The non-fluorescent cyanine dyes of the present invention are employedas acceptor dyes in assay methods utilising fluorescence resonanceenergy transfer. When a non-fluorescent cyanine dye of the invention isin an energy transfer relationship with a fluorescent donor dye, thefluorescence emission of the donor is reduced through quenching by theacceptor. When resonance energy transfer is lost through separation ofthe fluorescent donor dye and the acceptor dye, the fluorescenceemission due to the donor dye is restored Effective non-fluorescentquenching dyes have a low efficiency for converting absorbed incidentlight into fluorescence and, as such, are unsuitable as fluorescentlabels where a high degree of sensitivity is required. The relativeeffectiveness of the non-fluorescent cyanine dyes as quencher dyes isillustrated in FIG. 1 which shows the relative fluorescence emission ofrepresentative non-fluorescent cyanine dyes of the invention comparedwith corresponding dyes of the same class and with similar absorptioncharacteristics in the visible region of the spectrum.

The intrinsic fluorescence of the cyanine dyes of the present invention,when they are employed as energy acceptors is preferably less than 10%of the fluorescence emission of the donor dye upon excitation at thedonor excitation wavelength and detection of emission at the donoremission wavelength. FIG. 3 shows the decrease in background signalobtainable by the use of Compound II and Compound XI as acceptor dyescompared with that of a standard matched fluorescent cyanine acceptordye (Cy5), when measured at the emission maxima of the donor dye. Thecontribution to background fluorescence is thus minimised by the use ofthe non-fluorescent cyanine dyes of the invention as quencher dyes.Moreover, the present dyes are designed such that their spectral overlapwith a fluorescent donor dye is maximised, thereby improving efficiencyof quenching.

The biological material can be a biological molecule which may becleaved into the two component parts; or the biological material maycomprise two components as hereinbefore defined which may be boundeither by covalent or non-covalent association.

The present invention therefore relates to a novel fluorogenic substrateand an assay method for the detection and measurement of the cleavage ofa molecule into two component parts, the first component being labelledwith a fluorescent donor dye in an energy transfer relationship with anon-fluorescent cyanine acceptor dye bound to the second component.

The assays may be performed according to the present invention in highthroughput screening applications, including those in which compoundsare to be screened for their inhibitory effects, potentiation effects,agonistic, or antagonistic effects on the reaction under investigation.Examples of such assays include, but are not restricted to, the cleavageof a peptide or protein by a protease and the cleavage of a DNA or RNAmolecule by a nuclease. In this assay format, the enzyme substrate(peptide or nucleic acid) will include a sequence whose structurecombines a fluorescent donor dye molecule with the non-fluorescentcyanine acceptor dye, attached to the substrate at either side of thesubstrate bond to be cleaved. The substrate joins the fluorescent donorand the acceptor moieties in close proximity. The intrinsic fluorescenceof the donor is reduced through quenching by the acceptor due toresonance energy transfer between the pair of dyes. Resonance energytransfer becomes insignificant when the distance between the donor andacceptor moieties is greater than about 100 Angstroms. Cleavage of thesubstrate results in the separation between donor and acceptor dyes andconcomitant loss of resonance energy transfer. The fluorescence signalof the donor fluorescent dye increases, thereby enabling accuratemeasurement of the cleavage reaction.

Briefly, an assay for the detection of proteolytic enzyme activity maybe configured as follows. A reaction mixture is prepared by combining aprotease enzyme and a fluorogenic substrate which combines a fluorescentdonor dye molecule with a non-fluorescent acceptor dye of formula (2)attached to the substrate at either side of the substrate bond to becleaved. A known or a putative protease inhibitor compound may beoptionally included in the reaction mixture. Typically the reaction isperformed in buffered solution and the reaction is allowed to proceed tocompletion. The progress of the reaction may be monitored by observingthe steady state fluorescence emission due to the fluorescent donor dye,which is recorded using a spectrofluorimeter.

Alternatively, the invention relates to an assay method for detectingand measuring binding, by covalent or non-covalent association, of onecomponent of a ligand/reactant pair with a second component of saidpair, said first component being labelled with a fluorescent donor dyeand said second component being labelled with a non-fluorescent cyanineacceptor dye. Such assays are conveniently categorised as one of twotypes.

i) The first category comprises equilibrium binding assays, in which onecomponent of a specific binding pair binds non-covalently to a secondcomponent of the specific binding pair. Such equilibrium binding assaysmay be applied to screening assays in which samples containing compoundsto be screened are tested for their effect upon the binding of the firstcomponent of the specific binding pair (either antagonistic oragonistic), to the second component. Either component may be labelledwith the donor dye or the acceptor dye. In the absence of binding, thelabelled components are too far apart for resonance energy transfer tooccur. Upon binding of one labelled component to its labelled specificbinding partner, the label moieties are brought into sufficiently closeproximity for energy transfer to occur between donor and acceptorspecies resulting in a quenching of the donor fluorescence and adecrease in donor fluorescent signal.

For example, the dyes used in the present invention can be used to labelprobes such as those described by Tyagi and Kramer (loc. cit.) for usein the detection and identification of unique DNA sequences or specificgenes in a complete DNA molecule or mixtures of nucleic acid fragments.One end of the nucleic acid probe is labelled with a fluorescent dye andat the other end with a non-fluorescent cyanine acceptor dye. In theabsence of specific target sequence, the fluorescent and quenchingspecies will be held sufficiently close for energy transfer to occur.Consequently, irradiation of the fluorophore by excitation light willgive reduced fluorescent signal. Interaction of the probe with aspecific target nucleic acid sequence causes a conformational change totake place in the probe, such that the fluorescent donor and acceptorbecome separated by distance. Excitation of the fluorophore will resultin a fluorescent signal which may be recorded using aspectrofluorimeter.

Alternatively, the equilibrium binding assay may employ a sandwich assayformat in which one component of a specific binding pair, such as afirst antibody, is coated onto the wells of a microtitre well plate.Following binding of an antigen to the first antibody, a secondantigen-specific antibody is then added to the assay mix, so as to bindwith the antigen-first antibody complex; In this format, either thefirst antibody or the antigen may be labelled with the donor dye and thesecond antibody labelled with the acceptor dye or vice versa. In theabsence of binding of the first antibody-antigen-second antibodycomplex, the labelled components are too far apart for resonance energytransfer to occur. Upon binding of the second antibody with the firstantibody-antigen complex, the label moieties are brought intosufficiently close proximity for energy transfer to occur between donorand acceptor species resulting in a quenching of the donor fluorescenceand a decrease in donor fluorescent signal. Fluorescence signal ismeasured and the concentration of antigen may be determined byinterpolation from a standard curve.

Examples of specific binding pairs include, but are not restricted to,antibodies/antigens, lectins/glycoproteins, biotin/(strept)avidin,hormone/receptor, enzyme/substrate or co-factor, DNA/DNA, DNA/RNA andDNA/binding protein. It is to be understood that in the presentinvention, any molecules which possess a specific binding affinity maybe employed, so that the energy transfer dyes of the present inventionmay be used for labelling one component of a specific binding pair,which in turn may be used in the detection of binding to the othercomponent.

ii) In the second category, the assay may comprise detection andmeasurement of the addition of a fluorescent donor dye labelled moiety(the reactant) in solution in the assay medium to a non-fluorescentacceptor dye-labelled moiety (the substrate) or vice versa, by covalentattachment mediated through enzyme activity. Examples of such assaysinclude, but are not restricted to, the joining of DNA or RNA moleculesto other nucleic acid molecules by ligases, the addition of a nucleotideto a DNA or RNA molecule by a polymerase and the transfer of a labelledchemical moiety from one molecule to another by a transferase such asacetyl transferase. A known or a putative enzyme inhibitor may beoptionally included in the reaction mixture. It is to be understood thatany two appropriate reactant and substrate moieties may be employed.Either of the donor or the acceptor dyes of the present invention may beused for labelling one moiety which in turn may be used in the detectionand measurement of the reaction with the substrate.

For example, in a DNA ligation assay, DNA molecules to be joined aremixed together in aqueous buffer containing ATP in the presence of a DNAligase. Following incubation, the DNA strands are covalently attached inthe correct configuration by the formation of standard phosphodiesterlinkages in both strands of the duplex. Upon joining the label moietiesare bought into sufficiently close proximity for energy transfer tooccur between donor and acceptor species resulting in a quenching of thedonor fluorescence and a signal decrease which is proportional to theamount of ligated product formed.

The invention also relates to labelling methods wherein the non-fluorescent cyanine dyes of structures (1) and (2) including at leastone reactive or functional group at the R¹-R⁷ positions covalently reactwith amino, hydroxyl, aidehyde, phosphoryl, carboxyl, sulphydryl orother reactive groups on target materials. Such target materialsinclude, but are not limited to the group consisting of antigen,antibody, lipid, protein, peptide, carbohydrate, nucleotides whichcontain or are derivatized to contain one or more of an amino,sulphydryl, carbonyl, hydroxyl and carboxyl, phosphate and thiophosphategroups, and oxy or deoxy polynucleic acids which contain or arederivatized to contain one or more of an amino, sulphydryl, carbonyl,hydroxyl and carboxyl, phosphate and thiophosphate groups, microbialmaterials, drugs and toxins.

The selection of suitable fluorescent donor and acceptor pairs isgenerally dependent on several factors.

i) Firstly, the donor and acceptor chromophores should have strongelectronic transitions in the near UV to near IR spectral range.

ii) Secondly, the donor and acceptor moieties should be in relativelyclose proximity with each other. Suitably the donor and acceptor speciesshould be in the range 10 -100 Angstroms.

iii) Thirdly, there should be a suitable overlap between the donoremission spectrum and absorption spectrum of the acceptor. The greaterthe overlap between donor emission spectrum and the excitation spectrumof the acceptor, the greater the energy transfer. Energy transfer canoccur between dyes which share minimal spectral overlap, but this isonly observed when the dyes are in close proximity.

Suitable fluorescent donor dyes that can be combined with thenon-fluorescent cyanine acceptor dyes to form energy transfer pairs forthe practice of the present invention include the well known reactiveanalogues of the fluorescein, rhodamine and cyanine dyes. Other lowmolecular weight fluorescent dyes may be selected from the derivativesof the bis-pyrromethine boron difluoride dyes, such as3,3′,5,5′-tetramethyl-2,2′-pyrromethine-1,1′-boron difluoride, soldunder the trademark BODIPY by Molecular Probes Inc. Particularlypreferred are the cyanine dyes.

Suitable fluorescein donor dyes include: 5- and 6-carboxyfluorescein and6-carboxy-4′,5′-dichloro-2′,7′-dimethoxyfluorescein. Suitable rhodaminedyes include: 6-carboxyrhodamine (Rhodamine 110), 5-carboxyrhodamine-6G(R6G-5 or REG-5), 6-carboxyrhodamine-6G (R6G-6 or REG-6),N,N,N′,N′-tetramethyl-6-carboxyrhodamine (TAMRA or TMR),6-carboxy-X-rhodamine (ROX). Suitable cyanine donor dyes include theCyDyes™: Cy3, Cy3.5, Cy5, Cy5.5 and Cy7. (CyDye and Cy are trademarks ofAmersham Pharmacia Biotech UK Limited.) Cyanine dyes suitable for use asthe donor component in the assay of the present invention are disclosedin U.S. Pat. No.5268486 (Waggoner et al), or the rigidised cyanine dyessuch as those disclosed in GB Patent No.2301832 (Waggoner et al).Alternatively the fluorescent donor species used as the donor componentmay be a fluorescence energy transfer dye cassette. Examples of suchfluorescence energy transfer dye cassettes are to be found in GB PatentNo.2301833 (Waggoner et al).

Table 2 below shows examples of fluorescent donor dyes and correspondingnon-fluorescent cyanine acceptor dyes which are suitable for use in themethods according to the present invention.

TABLE 2 Donor Acceptor Cy32-{5-[1-(5-carboxypentyl)-3,3-dimethyl-5-sulpho-1,3-dihydro-2H-indol-2-ylidene]-1,3-pentadienyl}-1-ethyl-3,3- Cy3.5dimethyl-5-nitro-3H-indolinium (Compound I) * Cy52-{5-[1-(5-carboxypentyl)-3,3-dimethyl-5-sulpho-1,3-dihydro-2H-indol-2-ylidene]-1,3-pentadienyl}-1-(3,5-dinitrobenzyl)-3,3-dimethyl-5-nitro-3H-indolinium (Compound II) *1-butyl-2-{5-[1-(5-carboxypentyl)-3,3-dimethyl-6-sulpho-1,3-dihydro-2H-benzo[e]indol-2-ylidene]-1,3-pentadienyl}-3,3-dimethyl-5-nitro-3H-indolinium (Compound III) *1-butyl-2-{7-[1-(5-carboxypentyl)3,3-dimethyl-5-sulpho-1,3-dihydro-2H-indol-2-ylidene]-1,3,5-heptatrienyl}-3,3-dimethyl-5-nitro-3H-indolinium (Compound IV) * Cy5.51-butyl-2-{7-[1-(5-carboxypentyl)3,3-dimethyl-5-sulpho-1,3-dihydro-2H-indol-2-ylidene]-1,3,5-heptatrienyl}-3,3-dimethyl-5-nitro-3H-indolinium (Compound IV) * * salt form, undefined.

The non-fluorescent cyanine dyes of formulae (1) and (2) may be preparedby a process comprising:

a) reacting a first compound having the formula (A):

 where X, Z¹, R¹, R³ and R⁵ are hereinbefore defined,

b) a second compound which is the same or different from the firstcompound and having the formula (B):

 where Y, Z² R², R⁴, R⁶ are hereinbefore defined, and

c) a third compound suitable for forming a linkage between the first andsecond compounds, wherein a), c) and b) are reacted either in a two-orsingle step process to form the compounds of formulae (1) and (2).

In the case of a two step synthesis, an intermediate dye compound isfirst formed by reacting an indolenine compound of structure (A) with acompound suitable for forming the linkage wherein the reaction isperformed in a suitable polar solvent such as ethanol or acetic acid. Inthe second stage, the intermediate dye is reacted with the secondindolenine compound of structure (B) in a medium such as pyridine,acetic acid and acetic anhydride at room temperature. Such reactionconditions are also suitable for the preparation of non-fluorescentcyanine dyes of the present invention by a one step process. Reagents c)which may be used for forming the linkage between the indoleninemoieties are those suitable for forming a polymethine chain. Reagentsand methods suitable for forming cyanine dyes containing polymethinelinkages will be well known to those skilled in the art and includetriethyl orthoformate, malondialdehyde bis-(phenylimine) hydrochlorideand N-{(5-phenylamino)-2,4-pentadienylidene}aniline hydrochloride. (Seefor example, Fry D. J, Cyanine Dyes and Related Compounds, in Rodd'sChemistry of Carbon Compounds, Elsevier 1977, page 369-422).

The non-fluorescent cyanine dyes of the present invention may be used tocovalently label a target material such as a component of the assaysystem as hereinbefore described. Covalent labelling using compounds ofthe present invention may be accomplished with a target having at leastone functional or reactive group as hereinbefore defined. The target maybe incubated with an amount of a compound of the present inventionhaving at least one of R¹-R⁷ that includes a reactive or functionalgroup as hereinbefore defined that can covalently bind with thefunctional or reactive group of the target material. The target materialand the compound of the present invention are incubated under conditionsand for a period of time sufficient to permit the target material tocovalently bond to the compound of the present invention.

BRIEF DESCRIPTION OF DRAWINGS

The invention is further illustrated by reference to the followingexamples and figures.

Figures

FIG. 1 illustrates the relative fluorescence emission of Cy3 andCompounds V and VII (FIG. 1a) and of Cy5 and Compounds IX and XI (FIG.1b) according to Example 12.

FIG. 2 illustrates nucleic acid hybridisation assays in which areduction in fluorescence signal is observed, resulting from the bindingof a Cy3-labelled oligonucleotide and its complementary oligonucleotidelabelled with a non-fluorescent Cy5 analogue (Compound II) and withDabcyl, according to Example 13.

FIG. 3 illustrates the effect of using non-fluorescent cyanine dyes asacceptor dyes on signal to noise increase (and background reduction)compared with a standard matched fluorescent cyanine acceptor dye (Cy5)in an oligonucleotide hybridisation study according to Example 14.

FIG. 4 illustrates the course of protease cleavage assays utilising thesubstrate, Cy5-Ala-Ala-Phe-Phe-Ala-Ala-Lys-Compound III (SEQ ID No. 1),in the presence and in the absence of pepsin, measured with respect totime and fluorescence intensity, according to Example 15

EXAMPLES Example 1 Preparation of2-{5-[1-(5-Carboxypentyl)-3,3-dimethyl-5-sulpho-1,3-dihydro-2H-indol-2-ylidene]-1,3-pentadienyl}-1-ethyl-3,3-dimethyl-5-nitro-3H-indolinium,Salt (Compound I)

i) 5-Nitro-2,3,3-trimethylindole

Sodium nitrate (3.84 g, 45.2 mmol) was dissolved in concentratedsulphuric acid (100 ml). After cooling in ice, this solution was addedto a solution of 2,3,3-trimethylindole (6.65 g, 41 .8 mmol) inconcentrated sulphuric acid (100 ml) such that the temperature wasmaintained in the range 0-5° C. The reaction was stirred at 0-5° C. for90 minutes after completing the addition, then allowed to warm to roomtemperature and stirred for a further 16 hours. The mixture was pouredonto ice (200 g) then made basic by the addition of 50% aqueous sodiumhydroxide solution to pH12 (pH paper). The crude product was collectedby filtration and washed with water until the washings were neutral(˜1000 ml). The off-yellow solid was dried at the pump, dissolved inethyl acetate (250 ml) and dried further (MgSO₄). The solution wasfiltered and the red filtrate was rotary evaporated to dryness. Thesolid was dissolved in chloroform:ethyl acetate (95:5, 30 ml) andpurified by silica flash chromatography. This gave a dark yellow solid,5.12 g, 25 mmol, 60% yield. UV analysis (methanol) showed a single peakwith λmax=300 nm. Mass spectrometry (MALDI-TOF with a gentisic acidmatrix) gave m/z=203.8 (for C₁₁H₁₂N₂O₂=204.23). ¹H NMR:δ=1.97 (s, 6H),2.95 (s, 3H), 8.58 (d, J=10 Hz, 1H), 8.70 (s, 1H), 8.78 (d, J=10 Hz,1H).

ii) 1-Ethyl-5-nitro-2,3,3-trimethylindolium iodide

5-Nitro-2,3,3-trimethylindole (518 mg, 2.54 mmol) and ethyl iodide (2.5ml, 4.85 g, 31 mmol, excess) were mixed and heated to reflux for 8hours. After cooling to room temperature the excess ethyl iodide wasremoved by a gentle nitrogen stream and the residue was dissolved inchloroform and filtered through a plug of silica. The silica was washedwith chloroform (1 volume) and the combined filtrates were rotaryevaporated to give the product as a yellow oil, (486 mg, 1.35 mmol, 53%yield).

iii) 1-(5-Carboxypentyl)-5-sulpho-2,3,3-trimethylindolium bromide

To 5-sulpho-2,3,3-trimethylindole (potassium salt) (5.0 g, 18 mmol) wasadded 6-bromohexanoic acid (10.56 g, 53.8 mmol) in 1,2-propanediol (15ml) and heated at 80° C. for 72 hours. The mixture was cooled anddiluted with water (60 ml) and a solution of NaOH (10% in water, 60 ml)with stirring. The product was purified using preparative HPLC (Dynamax,C₁₈ column, gradient of TFA/H₂O to TFA/MeCN) to yield a grey/pink solid(2.4 g, 28%).

iv)2-(4-Anilinobuta-1,3-dienyl)-1-(5-carboxypentyl)-3,3-dimethyl5-sulpho-indolium,salt

To the product from stage iii) (400 mg, 0.85 mmol) was addedmalondialdehyde bis-(phenylimine) HCl (322 mg, 1 .24 mmol) in aceticacid (5 ml) and the mixture heated at 140° C. for 16 hours withstirring. The product was purified by HPLC (C₄ column, TFA/H₂O toTFA/MeCN gradient) to yield a red/grey solid (194.4 mg, 38%).

v) Synthesis of Compound (I)

1-Ethyl-5-nitro-2,3,3-trimethylindolium iodide (100 mg, 0.277 mmol) andthe product from stage iv) (50 mg, 0.104 mmol) were dissolved inpyridine:acetic acid:acetic anhydride (9:9:2, 5 ml) and the reaction wasstood at room temperature in the dark for 24 hours. After removing thesolvents by a gentle nitrogen stream the residue was dissolved inwater:acetonitrile (7:3, 5 ml) and the product was isolated by reversephase HPLC. UV (methanol): λmax abs=652 nm, λmax em=670 nm. MS:m/z=621.2 (for C₃₃H₄₀N₃O₇S=622.8).

Example 2 Preparation of2-{5-[1-(5-Carboxypentyl)-3,3-dimethyl-5-sulpho-1,3-dihydro-2H-indol-2-ylidene]-1,3-pentadienyl}-1-(3,5-dinitrobenzyl)-3,3-dimethyl-3H-indolinium,Salt (Compound II)

i) 1-(3,5-Dinitrobenzyl)-2,3,3-trimethylindolium iodide

3,5-Dinitrobenzyl chloride (100 mg, 0.46 mmol), 2,3,3-trimethylindole(74 μ, 73.2 mg, 0.46 mmol) and sodium iodide (69 mg, 0.46 mmol) wereheated at 100° C. in sulpholane (5 ml) for 16 hours. After cooling toroom temperature the product was isolated by reverse phase HPLC (89.7mg, 0.26 mmol, 57% yield).

ii)2-{5-[1-(5-Carboxypentyl)-3,3-Dimethyl-5-sulpho-1,3-dihydro-2H-indol-2-ylidene]-1,3-pentadienyl}-1-(3,5-dinitrobenzyl)-3,3-dimethyl-3H-indolinium,Salt (II)

N-(3,5-Dinitrobenzyl)-2,3,3-trimethylindolium iodide (20 mg, 0.059 mmol)and the product from Example 1 iv) (20 mg, 0.041 mmol) were dissolved inpyridine:acetic acid:acetic anhydride (2:2:1, 2.5 ml) and the reactionwas allowed to stand in the dark at room temperature for 16 hours. Theproduct was isolated by reverse phase HPLC to give 17.6 mg, 0.024 mmol,59% yield of compound (II). MS: m/z=730 (for C₃₈H₄₁N₄O₉S=729.8.)

iii)2{5[1-(5-Carboxypentyl)-3,3-Dimethyl-5-sulpho-1,3-dihydro-2H-indol-2-ylidene]-1,3-pentadienyl}-1-(3,5-dinitrobenzyl)-3,3-dimethyl-3H-indolinium,Salt, N-Hydroxysuccinimidyl Ester

O-(N-Succinimidyl)-N,N,N′,N′-bis-(tetramethylene)-uroniumhexafluorophosphate (10 mg, 0.04 mmol) was dissolved inN,N-dimethylformamide (1 ml) and N,N-diisopropylethylamine (11 μl, 0.063mmol) added. From this solution 100)1i was taken and added to thecarboxylic acid (1 mg) from ii) above. After 3hrs, mass spectrumanalysis indicated that the NHS ester had been formed. The product wasused without further purification for labelling. MS: m/z=797 (forC₄₃H₄₉N₄O₉S=797.9).

Example 3 Preparation of1-Butyl-2-{5-[1-(5-carboxypentyl)-3,3-dimethyl-6-sulpho-1,3-dihydro-2H-benzo[e]indol-2-ylidene[-1,3-pentadienyl}-3,3-dimethyl-5-nitro-3H-indolinium,Salt (Compound III)

1-Butyl-5-nitro-2,3,3-trimethylindolium iodide (2.45 mg, 9.374 μmol) wasadded to2-(2-anilinobutenyl)-1-(5-carboxypentyl)-3,3-dimethyl-6-sulpho-benz(e)indolium,salt (5 mg, 9.374 μmol) in pyridine:acetic acid:acetic anhydride (9:9:2;200 μ). The reaction mixture was stored at room temperature in the darkfor 4 days. The solvents were removed under reduced pressure and theresidue was purified by reverse phase HPLC to give the desired product(2.0 mg, 2.854 μmol, 30% yield).

Example 4 Preparation of1-Butyl-2-{7-[1-(5-carboxypentyl)3,3-dimethyl-5-sulpho-1,3-dihydro-2H-indol-2-ylidene]-1,3,5-heptatrienyl}-3,3-dimethyl-5-nitro-3H-indolinium,Salt (Compound IV)

i) 1-Butyl-5-nitro-2,3,3-trimethylindolium Iodide

5-Nitro-2,3,3-trimethylindole (250 mg, 1.22 mmol) and iodobutane (10 ml,16.17 g, 87.87 mmol) were mixed and heated to reflux for 16hrs. Thesolvent was removed and the impure material stored at−20° C. for use inlater reactions.

ii)1-Butyl-2-{7-[1-(5-carboxypentyl)3,3-dimethyl-5-sulpho-1,3-dihydro-2H-indol-2-ylidene)-1,3,5-heptatrienyl}-3,3-dimethyl-5-nitro-3H-indolium,Salt (IV)

1-(5-Carboxypentyl)-5-sulpho-2,3,3-trimethylindolium, salt (5 mg, 0.0141mmol), 1-butyl-5-nitro-2,3,3-trimethylindolium iodide (4.02 mg, 0.0141mmol) and N-[(5-phenylamino-)2,4-pentadienylidene]anilinemonohydrochloride (3.69 mg, 0.0141 mmol) were dissolved inpyridine:acetic acid:acetic anhydride (9:9:2, 1 ml) and the reaction wasallowed to stand at room temperature in the dark for 1 week. UV analysisat 752 nm over the week indicated the reaction was complete at the endof the week. The product was then isolated by reverse phase HPLC to givea blue solid (yield=4.3 mg, 45%). UV (methanol): λmax. abs=752 nm. MS:m/z=676 (for C₃₇H₄₆N₃O₇S=676.9).

Example 5 Preparation of2-{3-[1-(3-aminopropyl)-3,3-dimethyl-5-sulpho-1,3-dihydro-2H-indol-2-ylidene]-1-propenyl}-3-(3,5-dinitrobenzyl)-1,3-benzothiazol-3-ium,Salt (Compound V)

i) 3-(3,5-Dinitrobenzyl)-2-methyl-1,3-benzothiazol-3-ium Iodide

2-methylbenzothiazole (3 ml, 20.1 mmol) was dissolved in acetone (25ml), to which was added 3,5-dinitrobenzyl iodide (7.4 g, 24.1 mmol) andheated with stirring to reflux for 15 hours. The reaction was allowed tocool and added drop-wise to ether (500 ml) to produce a fine yellowsolid which was filtered and dried to constant weight in vacuo to give3-(3,5-dinitrobenzyl)-2-methyl-1,3-benzothiazol-3-ium iodide (5 g, 10.9mmol, 45% yield). MS: m/z=328 (C₁₅H₁₂N₃O₄S=330.3). ¹H NMR (200MHz-DMSO-d₆): δ 2.85 (s, 1 H); 3.7 (s, 3H); 6.35 (s, 2H); 7.37 (m, 2H);8.23 (d, 1H); 8.53 (d, 1H); 8.62 (s, 2H): 8.8 (s, 1H).

ii) 1-(3-Aminopropyl)-2,3,3-trimethyl-5-sulpho-3H-indolium Bromide

a) 2,3,3-Trimethylindolenium-5-sulphate (20 g, 84 mmol) andN-(3-bromopropyl)phthalimide (50 g, 186 mmols) were stirred insulpholane (75 ml) at 110° C. for 28 hours. Further amounts ofN-(3-bromopropyl)phthalimide (2.5 g, 84 mmol) were then added after 28hours and after 45 hours. The reaction mixture was heated for a total of65 hours then cooled and added to ethyl acetate (800 ml) forming aprecipitate which was filtered and washed with ethyl acetate (3×300 ml)and acetonitrile (5×400 ml) to give a pink solid which was dried invacuo to give 2,3,3-trimethyl-1-(3-phthalimidopropyl)-5-sulfo-indoliumbromide (25 g, 49 mmols, 59% yield).

b) 2,3,3-Trimethyl-1-(3-phthaiimidopropyl)-5-sulpho-indolinium bromide(14 g, 27.6 mmols) was dissolved in concentrated HCl (100 ml) withmethanol (50 ml). The reaction mixture was stirred at 100° C. for 32hrs.The reaction mixture was then cooled and the solvent removed underreduced pressure and dissolved in acetonitrile forming a precipitate.The solution was neutralised with concentrated ammonia (˜10 ml) and theprecipitate was filtered, dissolved in a minimum amount of water andpurified by running the solution through a 2 g plug of C18 silica. Thepink solid was freeze dried to give 1-(3-aminopropyl)-23,3-trimethyl-5-sulpho-3H-indolium bromide (8 g, 21.2 mmols, 77%yield).UV (methanol): λmax=254 nm. MS: m/z=294 (C₁₄H₂₁N₂O₃S=297). ¹H NMR (200MHz-DMSO-d₆):δ1.5 (2s, 6H); 2.1 (m, 2H); 2.98 (s, 3H); 3.3 (m, 2H,); 3.6(t, 2H); 6.6 (d, 1H, J=7.8 Hz); 7.5 (m, 2H); 8.2 (dd, 1H, J=4 and 6 Hz).

iii) Synthesis of Compound V

N,N′-Diphenylformamidine (236 mg, 1.2 mmol) was added to a solution of3-(3,5-dinitrobenzyl)-2-methyl-1,3-benzothiazol-3-ium iodide (500 mg,1.09 mmol) and 1-(3-aminopropyl)-2,3,3-trimethyl-5-sulpho-3H-indoliumbromide (412.5 mg, 1.09 mmol) dissolved in pyridine (4.5 ml), aceticacid (4.5 ml) and acetic anhydride (1 ml). The reaction was heated at100° C. for 3.5 hours then cooled to room temperature and purified byreverse phase HPLC. The pink solid isolated was then dissolved inaqueous hydrochloric acid (2.OM) and refluxed for 95 hours. The solutionwas then cooled to room temperature, evaporated and then re-purified byreverse phase HPLC. The pink solid collected was dried in vacuo. UV(ethanol): λmax=559 nm. MS: miz=638 (C₃₀H₃₀N₅S₂O₇=636.6).

Example 6 Preparation of2-[3-(3-(5-Carboxypentyl)-1,3-benzothiazol-2(3H)-ylidene)-1-propenyl]-3-(3,5-dinitrobenzyl)-1,3-benzothiazol-3-ium, Salt (CompoundVI)

i) 3-(5-Carboxypentyl)-2-methyl-1,3-benxothiazol-3-ium bromide

To 2-methylbenzothiazole (80 ml, 93.849, 0.629mol) was added6-bromohexanoic acid (250 g, 1.28mol). The reaction mixture was stirredat 140° C. for 24 hours. A yellow precipitate was formed. The reactionwas allowed to cool, dissolved in methanol (350 ml) and added dropwiseto ethyl acetate (2I). A beige precipitate was collected and washed withethyl acetate (3×50 ml). The solid was then dried to constant weight invacuo to give 3-(5-carboxypentyl)-2-methyl-1,3-benxothiazol-3-iumbromide (182.35 g 0.53mol, 84% yield). UV (methanol): λmax=237, 277 nm.MS: gave m/z=262 (C₁₄H₁₈NO₂₂S=264). ¹H NMR (200 MHz-DMSO-d₆):β1.49 (m,4H); 1.9 (m, 2H); 2.24 (t, 2HJ=6.35 Hz); 3.32 (s, 3H); 4.72 (t, 2HJ=7.81Hz); 7.85 (m, 2H); 8.4 (dd, 2H, J=8.31 Hz).

ii) 2-(2-Anilinoethenyl)-3-(3,5-dinitrobenzyl)-1,3-benzothiazol-3-ium,Salt

N,N′-Diphenylformamidine (42.9 mg, 0.29 mmol) was added to3-(3,5-dinitrobenzyl)-2-methyl-1,3-benzothiazol-3-ium iodide (100 mg,0.29 mmol), (prepared as in Example 5 i)) and sodium acetate (excess) inethanol (1 ml). The reaction was stirred at 40° C. for 4 hours to give ared solution. The solvent was removed under reduced pressure and theresidue was purified by reverse phase HPLC to yield an orange gum.

iii) Synthesis of Compound VI

3-(5-Carboxypentyl)-2-methyl-1,3-benzothiazol-3-ium bromide (7.9 mg,0.02 mmol) was added to2-(2-anilinoethenyl)-3-(3,5-dinitrobenzyl)-1,3-benzothiazol-3-ium salt(10 mg, 0.02 mmol) and sodium acetate (excess) in ethanol (1 ml). Thereaction was stirred at 45° C. and a pink solution was observed. Thesolvent was removed in vacuo and the residue was purified by reversephase HPLC to yield a dark pink solid. UV (water/acetonitrile): λmax=558nm. MS: m/z=604 (C₃₀H₂₇N₄O₆S₂=603.7).

Example 7 Preparation of3-[4-(carboxymethyl)benzyl]-2-{3-[1-(3,5-dinitrobenzyl)-3,3-dimethyl-5-sulpho-1,3-dihydro-2H-indol-2-ylidene]-1-propenyl}-1,3-benzoxazol-3-ium,Salt (Compound VII)

i) 3-[4-(Carboxymethyl)benzyl]-2-methyl-1,3-benzoxazol-3-ium Bromide

2-Methylbenzoxazole (yellow) was distilled under vacuum to give acolourless liquid (33 ml). 4-(Bromomethyl)phenylacetic acid (20 g, 87.3mmol) and 2-methylbenzoxazole (distilled, 33 ml, 278 mmol) weredissolved in 1,2-dichlorobenzene (70 ml). The reaction mixture washeated at 80° C. for 2.5 days to produce a thick yellow precipitate. Thereaction mixture was then cooled to room temperature, the precipitatecollected via filtration and washed with 1,2-dichlorobenzene (3×100 ml)and diethyl ether (4×200 ml). The cream solid collected was dried invacuo to give 3-(4-(carboxymethyl) benzyl]-2-methyl-1,3-benzoxazol-3-iumbromide (20 g, 55.3 mmol, 63% yield). UV (methanol): λmax=277 nm. MS:m/z=282 (C₁₇H₁₆NO₃=282.32). Normal phase TLC (ether) indicated that thestarting 2-methylbenzoxazole had an R_(f)=0.9±0.05 and the product anR_(f)=0. ¹H NMR (200 MHz-DMSO-d₆): δ1.75 (s, 2H); 3.2 (s, 3H); 5.85 (s,2H); 7.31 (d, 2H, J=6.8 Hz); 7.53 (d, 2H, J=6.8 Hz); 7.72 (m, 2H, J=6.3Hz); 7.89 (d, 1H, J=8.3 Hz); 8.17 (d, 1H, J=7.3 Hz); 9.8 (s, 1H).

ii) 1-(3,5-Dinitrobenzyl)-5-sulpho-2,3,3-trimethyl-3H-indolium Bromide

3,5-Dinitrobenzyl chloride (22.1g, 102 mmol) and sodium bromide (10.5 g,102 mmol) were added to 2,3,3-trimethyl-5-sulpho-indole, potassium salt(5.0 g, 20.4 mmol) in sulpholane (25 ml). The reaction mixture wasstirred at 100° C. for 5 hours. The solution was then cooled to roomtemperature and acetone (300 ml) was added to the reaction flask. Abrown precipitate was formed which was collected by filtration and thenpurified by reverse phase HPLC. This gave a beige solid which was driedin vacuo. UV (water/acetonitrile): λmax=283 nm. MS: m/z=420(C₁₈H₁₈N3O₇S=420.4).

iii)2-(2-Anilinoethenyl)-3-[4-(carboxymethyl)benzyl]-1,3-benzoxazol-3-ium,Salt

Ethyl N-phenylformimidate (0.94 g, 6.27 mmol) was added to3-[4-(carboxymethyl)benzyl]-2-methyl-1,3-benzoxazole-3-ium bromide (1.0g, 2.76 mmol) dissolved in 2-methoxyethanol (10 ml) and heated at 100°C. for 1.5 hours. The solution was then cooled to room temperature andpurified by reverse phase HPLC. The bright yellow solid obtained wasdried in vacuo.

iv) Synthesis of Compound VII

1-(3,5-Dinitrobenzyl)-2,3,3-trimethyl-5-sulpho-3H-indolium bromide (130mg, 0.26 mmol) was added to2-(2-anilinoethenyl)-3-[4-(carboxymethyl)benzyl]-1,3-benzoxazol-3-iumbromide (100 mg, 0.26 mmol) dissolved in pyridine and acetic anhydride(20:0.6, 10 ml). The reaction was kept in the dark for 24 hours at roomtemperature. The solvent was then removed and the product was isolatedafter purification by reverse phase HPLC. UV (ethanol): λmax=512 nm. MS:m/z=714 (C₃₆H₃₁N₄O₁₀S=711.7).

Example 8 Preparation of1-(5-Carboxypentyl)-2-f3-(1-(5-carboxypentyl)-3-methyl-5-nitro-1,3-dihydro-2H-benzimidazole-2-ylidene-1-propenyl)-3-methyl-5-nitro-3H-benzimidazol-1-ium,Salt (Compound VIII)

i) 1-(5-Carboxypentyl)-2,3-dimethyl-5-nitro-3H-benzimidazol-1-ium iodide

6-lodohexanoic acid (6.33 g, 26.15 mmol) was added to2,3-dimethyl-5-nitrobenzimidazole (1.0 g, 5.23 mmol) dissolved insulpholane (10 ml) and stirred for 24 hours at 100° C. The reactionmixture was then cooled to room temperature and added dropwise to ethylacetate (200 ml). A yellow precipitate was formed which was collected byfiltration and dried in vacuo. This gave1-(5-carboxypentyl)-2,3-dimethyl-5-nitro-3H-benzimidazol-1-ium iodide asa yellow solid (1.77 g, 4.09 mmol, 78% yield). UV (ethanol): λmax=285nm. MS: m/z=306 (C₁₅H₂₀N₃O₄=306). Normal phase TLC (ethanol) indicatedthe starting material with R_(f)=0.6±0.05 and the product with R_(f)=O.¹H NMR (200 MHz-DMSO-d₆):β 2.3 (m, 8H); 2.95 (s, 3H); 4.0 (s, 3H); 4.6(t, 2H); 8.24 (d, 1H, J=9.28 Hz); 8.5 (dd, 1H, J=9.28 and 1.95 Hz); 9.09(d, 1H, J=1.95 Hz).

ii)2-(2-Anilinoethenyl)-1-(5-carboxypentyl)-3-methyl-5-nitro-3H-benzimidazol-1-ium,salt

Ethyl N-phenylformimidate (384.9 mg, 2.58 mmol) was added to1-(5-carboxypentyl)-2,3-dimethyl-5-nitro-3H-benzimidazol-1-ium iodide(500 mg, 1.29 mmol) dissolved in 2-methoxyethanol (5 ml). The reactionwas heated at 100° C. for 1 hour, cooled to room temperature andpurified by reverse phase HPLC. The yellow solid was dried in vacuo forreacting further.

iii) Synthesis of Compound VIII

1-(5-Carboxypentyl)-2,3-dimethyl-5-nitro-3H-benzimidazol- 1 -ium iodide(9.4 mg, 0.024 mmol) was added to2-(2-anilinoethenyl)-1-(5-carboxypentyl)-3-methyl-5-nitro-3H-benzimidazol-1-liumsalt (10 mg, 0.024 mmol) dissolved in pyridine, acetic anhydride andtriethylamine (20:0.6:0.7, 2 ml). The reaction was kept in the dark for24 hours. The solvent was then evaporated and the product was isolatedafter purification by reverse phase HPLC. UV: λmax=535 nm. MS: m/z=622(C₃₁H₃₇N₆O₈=621.6).

Example 9 Preparation of2-{5-[1-(5-Carboxypentyl)-3,3-dimethyl-5,7-disulpho-1,3-dihydro-2H-benz[o]indol-2-ylidene]-1,3-pentadienyl}-3-(3,5-dinitrobenzyl)-1,3-benzothiazol-3-ium,Salt (Compound IX)

i) 1(5-Carboxypentyl)-2,3,3-trimethyl-5,7-disulpho-3H-benzo[e]indoliumbromide

6-Bromohexanoic acid (30 g, 0.153mol) was added to2,3,3-trimethyl-3H-benzo[e]indole-5,7-disulphonic acid (50 g, 0.112 mol)in nitrobenzene (200 ml) and stirred for 24 hours at 120° C. Thereaction mixture was cooled to room temperature. A brown precipitate wasformed which was collected by filtration and washed with 2-propanol (250ml). The solid was then recrystallised from 2-propanol. The taupe solidwas filtered and purified by reverse phase HPLC. This gave a grey solidwhich was dried in vacuo. UV (water/acetonitrile): λmax=273 nm, 283 nm.MS: m/z=484 (C₂₁H₂₆NO₈S₂=484.5). ¹H NMR (200 MHz-DMSO-d₆): δ 1.7 (m,14H); 2.2 t, 2H); 2.9 (s, 2H); 8.2 (d, 1H, J=9.28 Hz); 8.4 (d, 2H,J=4.39 Hz), 9.15 (d, 1H, J=9.28 Hz).

ii) Synthesis of Compound IX

Malonaldehydebisphenylimine monohydrochloride (141 mg, 0.55 mmol) wasadded to1-(5-carboxypentyl)-2,3,3-trimethyl-5,7-disulpho-3H-benzo[e]indoliumbromide (286 mg, 0.55 mmol) and3-(3,5-dinitrobenzyl)-2-methyl-1,3-benzthiazole-3-ium iodide (200 mg,0.55 mmol), (prepared as in Example 5 i)) dissolved in pyridine (18 ml),acetic acid (18 ml) and acetic anhydride (4 ml). The reaction wasstirred at 80° C. for 4 hours and cooled to room temperature. Thesolvent was evaporated and the mixture was purified by reverse phaseHPLC. The product, obtained as a blue solid, was dried in vacuo. UV(ethanol): λmax=676 nm. MS: m/z=853 (C₃₉H₃₇N₄O₁₂S₃=849.9).

Example 10 Preparation of1-Butyl-2-[7-(3-(5-carboxypentyl)-1,3-benzothiazol-2-ylidene)-1,3,5-heptatrienyl]-3,3-dimethyl-5-nitro-3H-indolenium,Salt (Compound X)

i) Synthesis of Compound X

N-[5-(Phenylamino)-2,4-pentadienylidene]aniline monohydrochloride (18mg, 0.064 mmol) was added to 1-butyl-2,3,3-trimethyl-5-nitro-3H-indoliumiodide (25 mg, 0.064 mmol) and3-(5-carboxypentyl)-2-methyl-1,3-benzothiazol-3-ium bromide (22 mg,0.064 mmol} in acetic acid (0.5 ml) and pyridine (0.5 ml). The reactionwas heated at 40° C. for 4 hours. The solution was then cooled to roomtemperature and the solvent evaporated under reduced pressure. The darkgreen residue was dissolved in dimethyl sulphoxide and purified byreverse phase HPLC to yield a green solid. UV (methanol): λmax=768 nm.MS: m/z=587 (C₃₄H₄₀N₃O₄S=586.7).

Example 11 Preparation of2-{5-[1-(5-Carboxypentyl)-3,3-dimethyl-5-sulpho-1,3-dihydro-2H-indol-2-ylidene]-1,3-pentadienyl}-1-(3,5-dinitrobenzyl)-3,3-dimethyl-5-sulfo-3H-indolium,Salt (Compound XI)

i) Synthesis of Compound XI

The reaction was carried out by an analogous method to that reported forCompound IX using1-(3,5-dinitrobenzyl)-2,3,3-trimethyl-5-sulfo-3H-indolium bromide (50mg, 0.12 mmol) and1-(5-carboxypentyl)-2,3,3-trimethyl-5-sulfo-3H-indolium bromide (43 mg,0.12 mmol) in the presence of malonaldehydebisphenyliminemonohydrochloride (32 mg, 0.12 mmol) in acetic acid (0.9 ml), pyridine(0.9 ml) and acetic anhydride (0.2 ml). The product was isolated as ablue solid. UV (ethanol): λmax=651 nm. MS: m/z=811(C₃₈H₄₁N₄O₁₂S₂=809.9).

Example 12 Relative Fluorescence Emission of Non-fluorescent CyanineDyes

Compounds V, VII, IX and XI were prepared as solutions in ethanol(5×10⁵M). For comparison, solutions of the fluorescent dyes Cy3 and Cy5were prepared in ethanol at the same concentration. The fluorescenceintensity of each of the dye solutions was measured using a fluorescenceplate reader. Serial dilutions (1:1) with ethanol of all dye solutionswere then made and the fluorescence intensity measured for eachdilution. The fluorescence emission of the non-fluorescent cyanine dyesrelative to the normal fluorescent cyanine dyes is shown in FIG. 1.

Example 13 Nucleic Acid Hybridisation Assay

13.1 Probe Preparation

Oligonucleotide A (5′-C6-amino modifier-TAC CCA GAC GAG CAA-biotin-3′)(SEQ ID No. 2) and the complementary oligonucleotide B (5′-TTG CTC GTCTGG GTA-C7-amino modifier-3′) (SEQ ID No. 3) were synthesised on anApplied Biosystems 391 DNA synthesiser using standard methods andmaterials. The oligonucleotides were deprotected for 17 hours at 40° C.and purified by reverse phase HPLC using C18 column and a 40%TEAA/acetonitrile gradient. The desired peaks were collected, freezedried and the samples were resuspended in sterile H₂O.

Oligonucleotides A and B were incubated with a 10-fold molar excess ofCy3 NHS-ester dye and2-{5-[1-(5-carboxypentyl)-3,3-dimethyl-5-sulpho-1,3-dihydro-2H-indol-2-ylidene]-1,3-pentadienyl}-1-(3,5-dinitrobenzyl)-3,3-dimethyl-5-nitro-3H-indoliniumsalt (Compound II)-NHS-ester respectively, in 0.1 M sodium bicarbonatebuffer (pH9), overnight at 22° C. The following morning, theoligonucleotides were precipitated using ethanol and the resultingpellets were resuspended in water. Labelled oligonucleotides werepurified by reverse phase HPLC using a C18 column and a 60%TEAA/acetonitrile gradient and the desired peaks collected and freezedried. Residues were resuspended into H20 and concentration of recoveredmaterial was determined.

A control oligonucleotide C (5′-TTG CTC GTC TGG GTA-Dabcyl-3′) (SEQ IDNo. 3) was synthesised as above. The DABCYL moiety was added using a3′-Dabcyl cpg column. Following synthesis, oligonucleotide C wasdeprotected for 17 hours at 40° C. and purified by HPLC as described.

13.2 Binding Assay

Wells of a black, streptavidin-coated 96-well plate were coated withCy3-labelled oligonucleotide A (100 pmol/well diluted in 100 μlPBS/1MgCl₂) for 120 minutes at ambient temperature. Any unbound materialwas removed by washing wells vigorously with buffer (PBS/1 mM MgCl₂/0.1%BSA). The Compound II-labelled oligonucleotide B and Dabcyl-labelledoligonucleotide C were diluted to 0.2 pmol/μl in buffer, and 100μl wasincubated with the coated wells at ambient temperatures for 120 minutes.Wells were washed vigorously with PBS and fluorescence intensity wasmeasured on a fluorescence plate reader using filter sets appropriatefor Cy3. Signals from wells coated with Cy3-labelled oligonucleotide Aalone were compared with those coated with Cy3-oligonucleotide A andincubated with either Compound II-labelled oligonucleotide B or controloligonucleotide C. The results are shown in FIG. 2. Wells coated withCy3-oligonucleotide A, gave a strong fluorescence signal. This signalwas reduced by 95% following hybridisation with Compound II-labelledoligonucleotide B and by 75% with oligonucleotide C.

Example 14 Use of Non-fluorescent Cyanine Dyes Compared with a StandardMatched Fluorescent Cyanine Acceptor Dye (Cy5) in an OligonucleotideHybridisation Study

An oligonucleotide (5′-TAC-CCA-GAC-GAG-CAA-3′) (SEQ ID No. 2), labelledat the 3′-terminus with biotin and at the 5′-terminus with Cy3, wasbound to the wells of a microtitre plate coated with streptavidin. Thecoated wells were then incubated with complimentary oligonucleotideprobes labelled individually with non-fluorescent cyanine acceptor dyes(Compound II and Compound XI). Following hybridisation, the wells werewashed and the relative fluorescence measured upon excitation of the Cy3donor dye. Control measurements were made on complementary unlabelled-,CyS-labelled and 2,4-dinitrobenzoyl-labelled oligonucleotides. Theresults are shown in FIG. 3. The data indicates that the use of thenon-fluorescent cyanine dyes (Compound II and Compound XI) is able toincrease the signal to noise ratio by approximately a factor of 2 overthat obtained with Cy5.

Example 15 Protease Cleavage Assay

15.1 Preparation of Protease Substrate

The peptide Ala-Ala-Phe-Phe-Ala-Ala-Lys (SEQ ID No. 1) was synthesisedon a Perkin Elmer 433A peptide synthesizer using standard Fmocchemistry. Cy5-NHS-ester dye (in slight molar excess) was coupled to theN-terminus of the peptide on the resin during overnight incubation inDMSO containing 2% v/v diisopropyl-ethylamine. The labelled peptideresin was washed sequentially with DMSO (˜10 ml), methanol (˜10 ml) anddichloromethane (˜5 ml) and dried. A 90 minute incubation in 95%TFA/2.5%triisopropylsilane/2.5% H₂0 facilitated side-chain deprotection andcleavage from the resin. After filtering through glass wool the peptidewas isolated as a blue precipitate in ice-cold diethylether. The productwas re-suspended in DMSO and purified by reverse-phase HPLC (gradient:water+0.1%TFA to 70% MeCN+0.1%TFA), the desired fractions beingcollected and freeze-dried.

Cy5-Ala-Ala-Phe-Phe-Ala-Ala-Lys (SEQ ID No. 1) was incubated with1-butyl-2-{5-[1-(5-carboxypentyl)-3,3-dimethyl-6-sulpho-1,3-dihydro-2H-benzo[e]indol-2-ylidene]-1,3-pentadienyl}-3,3-dimethyl-5-nitro-3H-indoliniumsalt (Compound III), NHS-ester, in slight molar excess in DMSOcontaining 2% v/v diisopropylethylamine overnight. The dual-labelledpeptide was again isolated by reverse-phase HPLC and freeze-dried. Theresidue was re-suspended into H₂O and the concentration determined.

15.2 Assay for Protease Enzyme

The protease substarte (Cy5-Ala-Ala-Phe-Phe-Ala-Ala-Lys-Compound III)Seq ID No 1) was diluted to 0.02 absorbance units/cm (at 650 nm) incitrate buffer (100 mM pH 3.0). To the solution (1 ml) of the labelledsubstrate in a cuvette, 25ng pepsin (10 μl volume) was added. Thefluorescence baseline prior to protease addition and the subsequentincrease in intensity were recorded at appropriate wavelengths. Acontrol experiment (minus pepsin) was performed in a similar manner. Theresults are shown in FIG. 4. Efficient quenching of Cy5 fluorescence inthe intact substrate results in a low signal. Protease-catalysedhydrolysis of the substrate removes this quenching, restoring the Cy5fluorescence. The increase in fluorescence intensity can be continuouslymonitored and is proportional to protease activity.

3 1 7 PRT artificial sequence synthetic peptide 1 Ala Ala Phe Phe AlaAla Lys 1 5 2 15 DNA artificial sequence synthetic oligonucleotide 2tacccagacg agcaa 15 3 15 DNA artificial sequence syntheticoligonucleotide 3 ttgctcgtct gggta 15

What is claimed is:
 1. A compound having the formula:

wherein the linker group Q contains at least one double bond and forms aconjugated system with the rings containing X and Y; groups R³, R⁴, R⁵and R⁶ are attached to the rings containing X and Y, or optionally, areattached to atoms of the Z¹ and Z² ring structures; Z¹ and Z² eachrepresent a bond or the atoms necessary to complete one or two fusedaromatic rings each ring having five or six atoms, selected from carbonatoms and, optionally, no more than two oxygen, nitrogen and sulphuratoms; X and Y are the same or different and are selected from bis-C₁-C₄alkyl- and C₄-C₅ spiro alkyl-substituted carbon, oxygen, sulphur,selenium, —CH═CH—and N-W wherein N is nitrogen and W is selected fromhydrogen, a group —(CH₂)_(m)R⁸ where m is an integer from 1 to 26 and R⁸is selected from hydrogen, amino, aldehyde, acetal, ketal, halo, cyano,aryl, heteroaryl, hydroxyl, sulphonate, sulphate, carboxylate,substituted amino, quaternary ammonium, nitro, primary amide,substituted amide, and groups reactive with amino, hydroxyl, carbonyl,carboxyl, phosphoryl, and sulphydryl groups; at least one of groups R¹,R², R³, R⁴ ₁, R⁵, R⁶ and R⁷ is the group —E—F where E is a spacer grouphaving a chain from 1-60 atoms selected from the group consisting ofcarbon, nitrogen, oxygen, sulphur and phosphorus atoms and F is a targetbonding group; any remaining groups R³, R⁴, R⁵, R⁶ and R⁷ groups areindependently selected from the group consisting of hydrogen, C₁-C₄alkyl, OR⁹, COOR⁹, nitro, amino, acylamino, quaternary ammonium,phosphate, sulphonate and sulphate, where R⁹ is selected from H andC₁-C₄ alkyl; any remaining R¹ and R² are selected from C₁-C₁₀ alkylwhich may be unsubstituted or substituted with phenyl the phenyl beingoptionally substituted by up to two substituents selected from carboxyl,sulphonate and nitro groups; characterised in that at least one of thegroups R¹, R², R³, R⁴, R⁵, R⁶ and R⁷ comprises at least one nitro groupwhich reduces the fluorescence emission of said compound such that it isessentially non-fluorescent; provided that the linker group Q is not asquaraine ring system.
 2. The compound according to claim 1 wherein Qcontains 1, 2 or 3 double bonds in conjugation with the rings containingX and Y.
 3. A compound having the formula:

wherein groups R³, R⁴, R⁵ and R⁶ are attached to the rings containing Xand Y or, optionally, are attached to atoms of the Z¹ and Z² ringstructures and n is an integer from 1-3; Z¹ and Z² each represent a bondor the atoms necessary to complete one or two fused aromatic rings eachring having five or six atoms, selected from carbon atoms and,optionally, no more than two oxygen, nitrogen and sulphur atoms; X and Yare the same or different and are selected from bis-C₁-C₄ alkyl- andC₄-C₆ spiro alkyl-substituted carbon, oxygen, sulphur, selenium,—CH═CH—and N-W wherein N is nitrogen and W is selected from hydrogen, agroup—(CH₂)_(m)R⁸ where m is an integer from 1 to 26 and R⁸ is selectedfrom hydrogen, amino, aldehyde, acetal, ketal, halo, cyano, aryl,heteroaryl, hydroxyl, sulphonate, sulphate, carboxylate, substitutedamino, quaternary ammonium, nitro, primary amide, substituted amide, andgroups reactive with amino, hydroxyl, carbonyl, carboxyl, phosphoryl,and sulphydryl groups; at least one of groups R¹, R², R³, R⁴, R⁵, R⁶ andR⁷ is the group—E—F where E is a spacer group having a chain from 1-60atoms selected from the group consisting of carbon, nitrogen, oxygen,sulphur and phosphorus atoms and F is a target bonding group; anyremaining groups R³, R⁴, R⁵, R⁶ and R⁷ groups are independently selectedfrom the group consisting of hydrogen, C₁-C₄ alkyl, OR⁹, COOR⁹, nitro,amino, acylamino, quaternary ammonium, phosphate, sulphonate andsulphate, where R⁹ is selected from H and C₁-C₄ alkyl; any remaining R¹and R² are selected from C₁∝C₁₀ alkyl which may be unsubstituted orsubstituted with phenyl the phenyl being optionally substituted by up totwo substituents selected from carboxyl, sulphonate and nitro groups;characterised in that at least one of the groups R¹, R², R³, R⁴, R⁵, R⁶and R⁷ comprises at least one nitro group which reduces the fluorescenceemission of saiiik compound such that it is essentially non-fluorescent.4. The compound according to claim 1 wherein the spacer group E isselected from: —(CHR′)_(p)— —{(CHR′)_(q)—O—(CHR′)_(r)—,}_(s)——{(CHR′)_(q)—NR′—(CHR′)_(r)}_(s)— —{(CHR′)_(q)—(CH═CH)—(CHR′)_(r)}_(s)——{(CHR′)_(q)—Ar—(CHR′)_(r)—}_(s)— —{(CHR′)_(q)—CO—NR′—(CHR′)_(r)—}_(s)——{(CHR′)_(q)—CO—Ar—NR′—(CHR′)_(r)—}_(s)— where R′ is hydrogen, or C₁-C₄alkyl which may be optionally substituted with sulphonate, Ar isphenylene, optionally substituted with sulphonate, p is 1-20, preferably1-10, q is 1-5, r is 0-5 and s is 1-5.
 5. The compound according toclaim 1 wherein said target bonding group comprises a reactive group forreacting with a functional group on a target material, or a functionalgroup for reacting with a reactive group on a target material.
 6. Thecompound of claim 5 wherein said reactive group is selected fromcarboxyl, succinimidyl ester, sulpho-succinimidyl ester, isothiocyanate,imaleimide, haloacetamide, acid halide, hydrazide, vinylsulphone,dichlorotriazine and phosphoramidite.
 7. The compound of claim 5 whereinsaid functional group is selected from hydroxy, amino, sulphydryl,imidazole, carbonyl including aldehyde and ketone, phosphate andthiophosphate.
 8. The compound according to claim 1 wherein at least oneof the groups R³, R⁴, R⁵, R⁶ and R⁷ is a nitro group and/or at least oneof the groups R¹ and R² is a mono- or di-nitro-substituted benzyl group.9. A biological material labelled with the compound of claim
 1. 10. Abiological material which comprises two components one of which islabelled with a fluorescent dye which may act as a donor of resonanceenergy and the other with a non-fluorescent cyanine dye according toclaim 1 and which may act as an acceptor of resonance energy transferredfrom the donor.
 11. A biological material according to claim 9 selectedfrom the group consisting of antigen, antibody, lipid, protein, peptide,carbohydrate, nucleotides which contain or are derivatized to containone or more of an amino, sulphydryl, carbonyl, hydroxyl and carboxyl,phosphate and thiophosphate groups, and oxy or deoxy polynucleic acidswhich contain or are derivatized to contain one or more of an amino,sulphydryl, carbonyl, hydroxyl and carboxyl, phosphate and thiophosphategroups, microbial materials, drugs and toxins.
 12. A method forlabelling a biological material comprising: i) adding to a liquid whichcontains a biological material selected from the group consisting of anantigen, antibody, lipid, protein, peptide, carbohydrate, nucleotides,microbial materials, drugs, toxins and combinations thereof a compoundaccording to claim 1, ii) reacting said compound with said biologicalmaterial wherein said compound covalently binds to and labels saidbiological material.
 13. An assay method which comprises: i) separatingtwo components which are in an energy transfer relationship, the firstcomponent being labelled with a fluorescent donor dye and the secondcomponent being labelled with a non-fluorescent cyanine acceptor dyeaccording to claim 1 and, ii) detecting the presence of the firstcomponent by measuring emitted fluorescence.
 14. A method according toclaim 13 wherein the assay is selected from proteolytic enzyme cleavageassays and nuclease enzyme cleavage assays.
 15. An assay method whichcomprises: i) binding one component of a specific binding pair with asecond component of said pair, said first component being labelled witha fluorescent donor dye and said second component being labelled with anon-fluorescent cyanine acceptor dye according to claim 1 so as to bringabout an energy transfer relationship between said first and secondcomponents; and, ii) detecting the binding of the first and secondcomponents by measuring emitted fluorescence.
 16. A method according toclaim 15 wherein said specific binding pair is selected from the groupconsisting of antibodies/antigens, lectins/glycoproteins,biotin/(strept)avidin, hormone/receptor, enzyme/substrate or co-factor,DNA/DNA, DNA/RNA and DNA/binding protein.
 17. A method according toclaim 15 wherein said binding assay is selected from the groupconsisting of immunoassays, nucleic acid hybridisation assays, proteinbinding assays, hormone receptor binding assays and enzyme assays.
 18. Abiological material according to claim 10 selected from the groupconsisting of antigen, antibody, lipid, protein, peptide, carbohydrate,nucleotides which contain or are derivatized to contain one or more ofan amino, suphydryl, carbonyl, hydroxyl and carboxyl, phosphate andthiophosphate groups, and oxy or deoxy polynucleic acids which containor are derivatized to contain one or more of an amino, suphydryl,carbonyl, hydroxyl and carboxyl, phosphate and thiophosphate groups,microbial materials, drugs and toxins.